Validation of a new technique to assess exhaled hydrogen peroxide: results from normals and COPD patients

Chronic airways inflammation in chronic obstructive pulmonary disease (COPD) induces the activation of several cell types with delivery of proteases and reactive oxygen species (ROS). Assessing oxidant content in the exhaled air of COPD patients has proven useful in monitoring airway inflammation. The present study was designed to confirm the usefulness of exhaled hydrogen peroxide concentration determination in COPD patients using a new technique which allows longer storage of the expired air condensate before the H2O2 assay. The technique was applied in 13 healthy nonsmoking subjects (six male, age range 22-40 yrs) and in seven patients (five male, age range 58-81 yrs) with mild or moderate COPD. Subjects breathed into a one-valve mouthpiece, and the exhaled air was directed into a vial kept at 0 degree C. After approximately 15 min of quiet breathing, 1 mL of expired air condensate was collected. An aliquot, 450 microL, of this sample was immediately added to an equal volume of a reaction mixture containing 2 mM 3,5,3',5'-tetramethylbenzidine and 40 microL of enzyme stock solution (0.5 mg.mL-1). After 15 min, 45 microL sulphuric acid was added (1 N final concentration), resulting in a reaction mixture pH of 1.0. After a further 10-min incubation, H2O2 concentration determination was performed spectrophotometrically at 450 nm. This solution, as well as the H2O2 assay, was stable for > or = 24 h if the sample was kept in the dark and at 4 degrees C. There was high stability on repeated measures, with a coefficient of variation equal to zero. The mean +/- SD H2O2 level in exhaled air from normal subjects was 0.12 +/- 0.09 microM, whereas it was significantly increased in COPD patients (0.50 +/- 0.11 microM; p = 0.0001 compared to healthy subjects). In three healthy control subjects, a normal H2O2 level in expired air increased to 0.70-0.80 microM during an acute upper respiratory tract infection. This new technique of hydrogen peroxide assay in expired air condensate greatly minimizes the inaccuracy deriving from the instability of hydrogen peroxide. The preliminary results obtained using this technique provide direct evidence for increased reactive oxygen species production in the airways of stable chronic obstructive pulmonary disease patients. However, the specificity of the procedure could be reduced by the interference of upper respiratory tract infections.